Rapamycin esters for treating transplantation rejection

ABSTRACT

A compound of the structure ##STR1## wherein R 1 , R 2 , and R 3  are each, independently, hydrogen or ##STR2##  with the proviso that R 1 , R 2 , and R 3  are not all hydrogen; R 4  is --(CH 2 ) m  X(CH 2 ) n  CO 2  R 5  or ##STR3## R 5  and R 6  are each, independently, alkyl, aralkyl, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl, alkoxy, hydroxy, cyano, halo, nitro, carbalkoxy, trifluoromethyl, amino, or a carboxylic acid; 
     X is ##STR4##  O, or S; R 7  and R 8  are each, independently, hydrogen or alkyl; 
     Y is CH or N; 
     m is 0-4; n is 0-4; 
     with the proviso that m and n are not both 0 when X is O or S; 
     or a pharmaceutically acceptable salt thereof, which is by virtue of its immunosuppressive activity is useful in treating transplantation rejection, host vs. graft disease, autoimmune diseases, and diseases of inflammation, by virtue of its antitumor activity useful in treating tumors, and by virtue of its antifungal activity is useful in treating fungal infections.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 07/777,983, now U.S. Pat.No. 5,221,670, filed Oct. 17, 1991 which is a continuation-in-part ofSer. No. 07/584,833, filed Sep. 19, 1990 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel esters of rapamycin and a method forusing them in the treatment of transplantation rejection, host vs. graftdisease, autoimmune diseases, diseases of inflammation, tumors, andfungal infections.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Seghal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmcol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989), rapamycin has been shown to be effective in inhibitingtransplant rejection (U.S. patent application Ser. No. 362,354 filedJun. 6, 1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183(1978).

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin(U.S. Pat. No. 4,650,803). Recently, the numbering convention forrapamycin has been changed; therefore according to Chemical Abstractsnomenclature, the esters described above would be at the 31- and 42-positions.

DESCRIPTION OF THE INVENTION

This invention provides derivatives of rapamycin which are useful asimmunosuppressive, anti-inflammatory, antitumor, and antifungal agentshaving the structure ##STR5## wherein R¹, R², and R³ are each,independently, hydrogen or ##STR6## with the proviso that R¹, R², and R³are not all hydrogen; R⁴ is --(CH₂)_(m) X(CH₂)_(n) CO₂ R⁵ or ##STR7## R⁵and R⁶ are each, independently, alkyl of 1-6 carbon atoms, aralkyl of7-10 carbon atoms, or phenyl which is optionally mono-, di-, ortri-substituted with a substituent selected from alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylicacid;

X is ##STR8## O, or S; R⁷ and R⁸ are each, independently, hydrogen oralkyl of 1-6 carbon atoms;

Y is CH or N;

m is 0-4;

n is 0-4;

with the proviso that m and n are not both 0 when X is O or S;

or a pharmaceutically acceptable salt thereof.

Of the compounds, preferred members are those in which R⁴ is --(CH₂)_(m)X(CH₂)_(n) CO₂ R⁵.

Aryl is defined as an organic radical derived from an aromatichydrocarbon by the removal of one atom; e.g., phenyl from benzene.Aralkyl is defined as an arylated alkyl radical; a radical in which analkyl H atom is substituted by an aryl group. The definition of aryl andaralkyl are also intended to encompass compounds in which the phenylgroups of such moieties are optionally mono-, di-, or tri-substitutedwith a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbonatoms, trifluoromethyl, amino, a carboxylic acid, or the like.

The pharmaceutically acceptable salts may be formed from inorganiccations such as sodium, potassium, and the like; mono-, di-, andtrialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, andtrihydroxyalkyl amines of 1-6 carbon atoms per alkyl group. Preferredsalts are formed from sodium cations and tris(hydroxymethyl)methylamine.

The compounds of this invention can be prepared by acylating rapamycinwith an acylating agent having the general structure ##STR9## where X isOH, in the presence of a coupling reagent, such asdicyclohexylcarbodimide. The compounds of this invention also can beprepared using an anhydride of the above described carboxylic acid asthe acylating species. Alternatively, the acylating species can be anacid halide, where X can be Cl, Br, or I. The acylating groups used toprepare the compounds of this invention are commercially available orcan be prepared by methods that are disclosed in the literature.

Immunosuppressive activity was evaluated in an in vitro standardpharmacological test procedure to measure lymphocyte proliferation (LAF)and in two in vivo standard pharmacological test procedures. The firstin vivo procedure was a popliteal lymph node (PLN) test procedure whichmeasured the effect of compounds of this invention on a mixed lymphocytereaction and the second in vivo procedure evaluated the survival time ofa pinch skin graft.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly, cells from the thymus of normalBALB/c mice are cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated; radioactivity isdetermined. Inhibition of lymphoproliferation is assessed in percentchange in counts per minute from non-drug treated controls. The resultsare expressed by the following ratio: ##EQU1##

A mixed lymphocyte reaction (MLR) occurs when lymphoid cells fromgenetically distinct animals are combined in tissue culture. Eachstimulates the other to undergo blast transformation which results inincreased DNA synthesis that can be quantified by the incorporation oftritiated thymidine. Since stimulating a MLR is a function of disparityat Major Histocompatibility antigens, an in vivo popliteal lymph node(PLN) test procedure closely correlates to host vs. graft disease.Briefly, irradiated spleen cells from BALB/c donors are injected intothe right hind foot pad of recipient C3H mice. The drug is given daily,p.o. from Day 0 to Day 4. On Day 3 and Day 4, tritiated thymidine isgiven i.p., b.i.d. On Day 5, the hind popliteal lymph nodes are removedand dissolved, and radioactivity counted. The corresponding left PLNserves as the control for the PLN from the injected hind foot. Percentsuppression is calculated using the non-drug treated animals asallogenic control. Rapamycin at a dose of 6 mg/kg, p.o. gave 86%suppression, whereas cyclosporin A at the same dose gave 43%suppression. Compounds evaluated in the PLN test procedure wereadministered orally, unless otherwise indicated, as being administeredintraperitoneally. Carboxymethyl cellulose was used as the vehicle foradministration, unless otherwise indicated. Results are expressed by thefollowing ratio, unless otherwise indicated: ##EQU2##

The second in vivo test procedure is designed to determine the survivaltime of pinch skin graft from male DBA/2 donors transplanted to maleBALB/c recipients. The method is adapted from Billingham R. E. andMedawar P. B., J. Exp. Biol. 28:385- 402, (1951). Briefly, a pinch skingraft from the donor is grafted on the dorsum of the recipient as ahomograft, and an autograft is used as control in the same region. Therecipients are treated with either varying concentrations of cyclosporinA as test control or the test compound, intraperitoneally. Untreatedrecipients serve as rejection control. The graft is monitored daily andobservations are recorded until the graft becomes dry and forms ablackened scab. This is considered as the rejection day. The mean graftsurvival time (number of days ±S.D.) of the drug treatment group iscompared with the control group.

The following table summarizes the results of representative compoundsof this invention in these three standard test procedures.

                  TABLE 1                                                         ______________________________________                                                 LAF*       PLN*         Skin Graft                                   Compound (ratio)    (ratio)      (days + SD)                                  ______________________________________                                        Example 1                                                                              0.37       ++            8.2 ± 1.2                                Example 2                                                                              0.9        0.69**       10.7 ± 1.2                                Example 3                                                                              3.27       1.04**       12.7 ± 0.9                                                    0.20                                                                          2.08                                                      Example 4                                                                              0.56       1.68         10.2 ± 1.7                                                    0.42                                                      Example 5                                                                              0.02       1.11          8.0 ± 1.7                                Example 6                                                                              0.01       0.48          8.0 ± 0.9                                Example 7                                                                              0.97       0.70**       12.0 ± 1.0                                Example 8                                                                              0.22       -1.93         9.3 ± 1.6                                                    0.37**,+                                                  Example 9                                                                              0.22       0.41         10.2 ± 1.2                                Example 10                                                                             0.18       0.39         10.8 ± 0.8                                Example 11                                                                             0.00       0.09          7.8 ± 1.7                                Example 12                                                                             97%+++     1.04         10.8 ± 0.4                                Example 13                                                                             2.11       1.02         10.6 ± 0.9                                                    0.40                                                      Rapamycin                                                                              1.0        1.0          12.0 ± 1.7                                ______________________________________                                         *Calculation of ratios was described supra.                                   **Administered using cremophore/ethanol as the vehicle.                       +Administered intraperitoneally.                                              ++Not evaluated.                                                              +++Result expressed as percent inhibition at 100 nM.                     

The results of these standard pharmacological test proceduresdemonstrate immunosuppressive activity both in vitro and in vivo for thecompounds of this invention. Positive ratios in the LAF and PLN testprocedures indicate suppression of T cell proliferation. As atransplanted pinch skin grafts are typically rejected within 6-7 dayswithout the use of an immunosuppressive agent, the increased survivaltime of the skin graft when treated with the compounds of this inventionfurther demonstrates their utility as immunosuppressive agents. While itappears that the compound disclosed by Example 8 may cause T cellproliferation in the PLN test procedure because of the -1.93 ratioobtained, it is believed that this result is merely an anomaly in lightof the other data obtained. Spurious results have been obtained in thePLN test procedure using compounds that have low bioavailability. Lowbioavailability can be due to the compound itself, the dose used, thevehicle, the route of administration, or a combination of any of theabove factors. When the negative ratio was obtained for the compound ofExample 8, it was administered orally in carboxymethylcellulose. Anegative ratio in the PLN test procedure was not observed for thecompounds of Examples 9 and 10, which are pharmaceutical salts of thecompound of Example 8. When the compound of Example 8 was administeredi.p. in a mixture of cremophore and ethanol as the vehicle, a positiveratio was obtained indicating the compound had immunosuppressiveactivity. The positive ratio obtained in the LAF test procedure coupledwith the increased survival time observed in the skin graft testprocedure confirm the immunosuppressive activity of the compound ofExample 8. The negative ratio obtained when the compound of Example 8was administered orally in carboxymethyl cellulose is therefore believedto be attributed to low bioavailability, and not a function of itsimmunosuppressive activity.

Antifungal activity of the compounds of this invention was measuredagainst 5 strains of Candida albicans using a plate test procedure formeasurement of inhibition. The following represents the typicalprocedure used. Compound to be tested was placed on sterile dried 1/4"plate disks, and allowed to dry. Agar plates were seeded with fungi andallowed to solidify. The impregnated disks were placed on the seededAgar surface and incubated for the time required for the particularculture. Results are expressed in MIC (μg/ml) to inhibit growth. Theresults of this test procedure showed that the compounds of thisinvention have antifungal activity; however, it was surprising that thecompounds of this invention were less active that the parent compound,rapamycin. The compounds of Examples 12 and 13 were not evaluated forantifungal activity, but because of the structural similarity to theones that were evaluated, they too are considered to have antifungalactivity.

                                      TABLE 2*                                    __________________________________________________________________________    Strain of Candida albicans                                                    Compound                                                                            ATCC 10231                                                                           ATCC 38246                                                                           ATCC 38247                                                                           ATCC 38248                                                                            3669                                       __________________________________________________________________________    Example 1                                                                           >0.4   >0.4   >0.4   >0.4    >0.4                                       Example 2                                                                           >0.4   0.4    >0.4   0.4     0.4                                        Example 3                                                                           0.2    0.1    0.4    0.1     0.1                                        Example 4                                                                           >0.4   0.2    >0.4   0.2     0.4                                        Example 5                                                                           0.4    >0.4   >0.4   >0.4    >0.4                                       Example 6                                                                           0.4    >0.4   0.4    >0.4    >0.4                                       Example 7                                                                           0.1    0.4    0.1    0.1     0.2                                        Example 8                                                                           0.4    >0.4   0.4    >0.4    >0.4                                       Example 9                                                                           0.2    >0.4   0.2    0.4     >0.4                                       Example 10                                                                          0.1    >0.4   0.2    0.4     >0.4                                       Example 11                                                                          >0.4   >0.4   >0.4   >0.4    >0.4                                       Rapamycin                                                                           0.003  0.025  0.003  0.006   0.025                                      __________________________________________________________________________     *expressed as MIC (μg/ml)                                             

Based on the results of these standard pharmacological test procedures,the compounds are useful in the treatment of transplantation rejectionsuch as, heart, kidney, liver, bone marrow, and skin transplants;autoimmune diseases such as, lupus, rheumatoid arthritis, diabetesmellitus, myasthenia gravis, and multiple sclerosis; diseases ofinflammation such as, psoriasis, dermatitis, eczema, seborrhea, andinflammatory bowel disease; and fungal infections. As the compounds ofthis invention are structurally related to rapamycin, which hasantitumor activity, they too are considered to be useful as antitumoragents.

The compounds may be administered neat or with a pharmaceutical carrierto a mammal in need thereof. The pharmaceutical carrier may be solid orliquid.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carder can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

Preferably, the pharmaceutical composition is in unit dosage form, e.g.as tablets or capsules. In such form, the composition is sub-divided inunit dose containing appropriate quantities of the active ingredient;the unit dosage forms can be packaged compositions, for example,packeted powders, vials, ampoules, prefilled syringes or sachetscontaining liquids. The unit dosage form can be, for example, a capsuleor tablet itself, or it can be the appropriate number of any suchcompositions in package form. The dosage to be used in the treatmentmust be subjectively determined by the attending physician.

In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles containing 0.1-5 percent, preferably 2%, of activecompound which may be administered topically

The following examples illustrate the preparation of representativecompounds of this invention.

EXAMPLE 1 Rapamycin-14,31,42-tris(monobenzylsuccinate)

To a solution of 5.0 g (5.47 mmol) of rapamycin, 3.41 g (16.41 mmol) ofmonobenzylsuccinate, and 3.15 g (16.41 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20 mL ofdry dichloromethane was added 200 mg of 4-dimethylaminopyridine. Thesolution was stirred at room temperature for 3 days. The reactionmixture was poured into 2N HCl and extracted three times with ethylacetate. The organic layers were combined, washed with brine, dried overanhydrous sodium sulfate, decanted, and concentrated in vacuo to give alight yellow foam. Flash chromatography on a 60 mm×150 mm silica gelcolumn eluting with 20% ethyl acetate/hexane to 75% ethyl acetate/hexanegave three fractions. Fraction #1, upon concentration, gave 330 mg(4.1%) of pure rapamycin-14,31,42-tris(monobenzylsuccinate).

¹ H NMR (CDCl₃, 400 MHz) δ7.353 (bs, 15H, arom), 5.168 (d, J=2.0 Hz, 1H,CH--O₂ C), 5.148 (m, 6H, CH₂ Ph), 4.672 (m, 1H, CO₂ CH--CHOMe), 3.355(s, 3H, CH₃ O--), 3.337 (s, 3H, CH₃ O--), 3.327 (s, 3H, CH₃ O--), 2.697(m, 12H, O₂ CCH₂ CH₂ CO₂ CH₂ Ph), 1.745 (s, 3H, CH₃ C═C), 1.655 (s, 3H,CH₃ C═C); IR (KBr) 3450 (OH), 2950 (CH), 1745 (C═O), 1650, 1460, 1385,1360, 1160, 1105, 995 cm⁻¹.

Analysis Calcd for C₈₄ H₁₀₉ NO₂₁.3H₂ O: C, 66.27; H, 7.56; N, 0.92.Found: C, 65.96; H, 7.24; N, 1.00.

The following representative compounds can be prepared from rapamycinand the appropriate half acid-ester by employing the method used toprepare the title compound in Example 1.

Rapamycin-14,31,42-tris (monomethylsuccinate)

Rapamycin-14,31,42-tris (monophenyl-3',3'-dimethylglutarate)

Rapamycin-14,31,42-tris (mono t-butyl-3'-methylglutarate)

Rapamycin-14,31,42-tris (monobenzylthiodiglycolate)

Rapamycin-14,31,42-tris (monohexyldiglycolate)

Rapamycin-14,31,42-tris (monopropylphthalate)

Rapamycin-14,31,42-tris (monoethyl-2',6'-pyridinedicarboxylate)

EXAMPLE 2 Rapamycin-31,42-bis(monobenzylsuccinate)

Fraction #2, obtained from the procedure employed in Example 1, gave1.25 g (17.7%) of pure rapamycin-31,42-bis(monobenzylsuccinate) uponconcentration.

1H NMR (CDCl₃, 400 MHz) δ7.351 (bs, 10H, arom), 5.168 (d, J=2.0 Hz, 1H,CH--O₂ C), 5.125 (m, 4H, CH₂ Ph), 4.680 (m, 1H, CO₂ CH--CHOMe), 3.356(s, 3H, CH₃ O--), 3.329 (s, 3H, CH₃ O--), 3.146 (s, 3H, CH₃ O--), 2.639(m, 8H, O₂ CCH₂ CH₂ CO₂ CH₂ Ph), 1.748 (s, 3H, CH₃ C═C), 1.654 (s, 3H,CH₃ C═C); IR (KBr) 3450 (OH), 2940 (CH), 1740 (C═O), 1650, 1455, 1380,1355, 1160, 1105, 995 cm⁻¹ ; MS (neg. ion FAB) 1294 (M--), 1202, 1103,1012, 590, 511, 475, 297, 207, 167, 148, 99 (100); High Res. MS (neg.ion FAB) Calcd for C₇₃ H₉₉ NO₁₉ 1293.68108, found 1293.6811.

Analysis Calcd for C₇₃ H₉₉ NO₁₉.H₂ O: C, 66.82; H, 7.70; N, 1.07. Found:C, 67.17; H, 7.67; N, 1.23.

The following representative compounds can be prepared from rapamycinand the appropriate half acid-ester by employing the method used toprepare the title compound in Example 2.

Rapamycin-31,42-bis (monomethylsuccinate)

Rapamycin-31,42-bis (monophenyl-3',3'-dimethylglutarate)

Rapamycin-31,42-bis (mono t-butyl-3'-methylglutarate)

Rapamycin-31,42-bis (monobenzylthiodiglycolate)

Rapamycin-31,42-bis (monohexyldiglycolate)

Rapamycin-31,42-bis (monopropylphthalate)

Rapamycin-31,42-bis (monoethyl-2',6'-pyridinedicarboxylate)

EXAMPLE 3 Rapamycin-42-(monobenzylsuccinate)

Fraction #3, obtained from the procedure employed in Example 1, gave 930mg (15.4%) of pure rapamycin-42-monobenzylsuccinate upon concentration.

¹ H NMR (CDCl₃, 400 MHz) δ7.355 (bs, 5H, arom), 5.141 (m, 2H, CH₂ Ph),4.680 (m, 1H, CO₂ CH--CHOMe), 3.364 (s, 3H, CH₃ O--), 3.333 (s, 3H, CH₃O--), 3.141 (s, 3H, CH₃ O--), 2.698 (m, 4H, O₂ CCH₂ CH₂ CO₂ CH₂ Ph),1.751 (s, 3H, CH₃ C═C), 1.655 (s, 3H, CH₃ C═C); IR (KBr) 3450 (OH), 2940(CH), 1740 (C═O), 1645, 1455, 1380, 1165, 1105, 990 cm⁻¹ ; MS (neg. ionFAB) 1103 (M--), 1045, 1012, 624, 590, 167, 99 (100); High Res. MS (neg.ion FAB) Calcd for C₆₂ H₈₉ NO₁₆ 1103.6181, found 1103.6048.

Analysis Calcd for C₆₂ H₈₉ NO₁₆.H₂ O: C, 66.36; H, 8.02; N, 1.24. Found:C, 66.02; H, 7.69; N, 1.26.

The following representative compounds can be prepared from rapamycinand the appropriate half acid-ester by employing the method used toprepare the title compound in Example 3.

Rapamycin-42-monophenyl-3',3'-dimethylglutarate)

Rapamycin-42-(mono t-butyl-3'-methylglutarate)

Rapamycin-42-(monobenzylthiodiglycolate)

Rapamycin-42-(monohexyldiglycolate)

Rapamycin-42-(monopropylphthalate)

Rapamycin-42-(monoethyl-2',6'-pyridinedicarboxylate)

EXAMPLE 4 Rapamycin-31,42-bishemiglutarate

To a solution of 2.0 g (2.2 mmol) of rapamycin in 10 mL of drydichloromethane was added 1.24 g (10.9 mmol) of glutaric anhydridefollowed by 881 uL (861 mg, 10.9 mmol) of pyridine. To this was added200 mg of 4-dimethylaminopyridine and the reaction mixture was allowedto reflux for 8 h. The solution was cooled to room temperature, pouredinto 2N HCl, and extracted three times with dichloromethane. Thecombined organic extracts were washed with brine, dried over anhydroussodium sulfate, decanted, and concentrated in vacuo to give a yellowfoam. The crude product was purified via reverse phase HPLC on a C₁₈column eluting starting with 60% acetonitrile/water. Collected, after,concentration, 586 mg (24%) of rapamycin-31,42-bishemiglutarate.

¹ H NMR (CDCl₃, 400 MHz) δ5.398 (m, 1H, --CO₂ CHCHOMe), 4.683, (m, 1H,--CO₂ CHCHOMe), 3.364 (s, 3H, CH₃ O--), 3.362 (s, 3H, CH₃ O--), 3.106(s, 3H, CH₃ O--), 2.407 (m, 8H, --O₂ CCH₂ CH₂ CH₂ CO₂ H), 1.960 (m, 4H,--O₂ CCH₂ CH₂ CH₂ CO₂ H), 1.770 (s, 3H, CH₃ C═C), 1.653 (s, 3H, CH₃C═C); ¹³ C NMR (CDCl₃, MHz) 211.45 (C═O), 206.84 (C═O), 200.44 (C═O),177.83 (C═O), 177.04 (C═O), 172.43 (C═O), 171.20 (C═O), 165.27 (C═O),159.08 (C═O); IR (KBr) 3430 (OH), 2940 (CH), 2880 (CH), 1745 (C═O),1685, 1625, 1580, 1450, 1385, 1330, 1200, 1140, 1100, 990 cm⁻¹ ; MS(neg. ion FAB) 1140 (M--H), 1122, 1026, 990, 946, 913, 590, 475, 435,321, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcd for C₆₁H₉₀ O₁₉ N (M--H) 1140.6107, Found 1140.6106.

Analysis Calcd for C₆₁ H₉₁ O₁₉ N.H₂ O: C, 63.15; H, 8.02; N, 1.20. FoundC, 63.35; H, 7.88; N, 1.40.

The following representative compounds can be prepared from rapamycinand the appropriate anhydride by employing the method used to preparethe title compound in Example 4.

Rapamycin-31,42-bishemi-3'-methylglutarate

Rapamycin-31,42-bishemi-3',3'-dimethylglutarate

Rapamycin-31,42-bishemi-3'-oxoglutarate

Rapamycin-31,42-bishemi-3'-thioglutarate

Rapamycin-31,42-bishemi-phthalate

Rapamycin-31,42-bishemi-2',3'-pyridine dicarboxylate

EXAMPLE 5 Rapamycin-31,42-hemiglutarate bissodium salt

Purified bis-31,42-hemiglutarate of rapamycin (740 mg, 649 umol),prepared as described in Example 4, was dissolved in 5 mL of 95% ethanoland 107 mg (1.27 mmol) of sodium bicarbonate was added. Water (1 mL) wasadded to completely dissolve the salt. Once dissolved, the light yellowsolution was concentrated in vacuo to give a foamy yellow solid. Thefoam was dried in a drying pistol for 24 h, refluxing over acetone atreduced pressure to give 520 mg of the bissodium salt.

¹ H NMR (d₆ -DMSO, 400 MHz) δ5.235 (m, 1H,--CHO₂ C), 4.498 (m, 1H,MeOCHCHO₂ C--), 3.287 (s, 6H, 2CH₃ O--), 3.236 (s, 3H, CH₃ O--), 2.245(m, 8H, O₂ CCH₂ CH₂ CH₂ CO₂ --), 1.712 (s, 3H, CH₃ C═C), 1.593 (s, 3H,CH₃ C═C); IR (KBr) 3420 (OH), 2920 (CH), 1725 (C═O), 1675, 1620, 1560,1450, 1400, 1375, 1230, 1195, 1130, 1090, 980 cm⁻¹ ; MS (neg. ion FAB)1112 (M--1, free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100);High Res. MS (neg. ion FAB) Calcd for C₆₁ H₈₉ O₁₉ NNa (M--Na) 1162.5926,Found 1162.5899.

Analysis Calcd for C₆₁ H₈₉ O₁₉ NNa₂.H₂ O: C, 60.85; H, 7.56; N, 1.16,Found: C, 60.67; H, 7.36; N, 1.58.

EXAMPLE 6 Rapamycin-31,42-bishemiglutarate bistromethamine salt

Purified bis-31,42 hemiglutarate of rapamycin (950 mg, 833 umol),prepared as described in Example 4, was dissolved in 5 mL of 95% ethanoland 197 mg (1.63 mmol) of tris(hydroxymethyl)methylamine was added.Water (1 mL) was added to completely dissolve the amine. Once dissolved,the yellow solution was concentrated in vacuo to give a foamy yellowsolid. The very hygroscopic foam was dried in a drying pistol for 24 h,refluxing over acetone at reduced pressure to give 900 mg (78%) of thebistromethamine salt.

¹ H NMR (d₆ -DMSO, 400 MHz) δ5.253 (m, 1H,--CHO₂ C), 4.523 (m, 1H,MeOCHCHO₂ C--), 3.347 (s, 6H, 2 CH₃ O--), 3.276 (s, 3H, CH₃ O--), 2.289(m, 8H, O₂ CCH₂ CH₂ CH₂ CO₂ --), 1.681 (s, 3H, CH₃ C═C), 1.595 (s, 3H,CH₃ C═C); IR (KBr) 3400 (OH), 2920 (CH), 1730 (C═O), 1620, 1555, 1450,1400, 1370, 1185, 1060, 980 cm⁻¹ ; MS (neg. ion FAB) 1140 (M--H, freeacid), 1028, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcdfor C₆₁ H₉₀ O₁₉ N (M--H, free acid) 1140.6107, Found 1140.6069.

Analysis Calcd for C₆₉ H₁₀₃ O₂₅ N₃.2H₂ O: C, 58.77; H, 7.58; N, 2.98.Found: C, 58.47; H, 7.94; N, 3.58.

EXAMPLE 7 Rapamycin-42-hemi-3'-oxoglutarate

To a solution of 3.0 g (3.3 mmol) of rapamycin in 20 mL of drydichloromethane was added 1.90 g (16.4 mmol) of diglycolic anhydridefollowed by 1.32 mL (1.29 g, 16.4 mmol) of pyridine. To this was added200 mg of 4-dimethylaminopyridine and the reaction mixture was allowedto stir at room temperature for 2 days. The solution was cooled to roomtemperature, poured into 2N HCl, and extracted three times withdichloromethane. The combined organic extracts were washed with brine,dried over anhydrous sodium sulfate, decanted, and concentrated in vacuoto give a yellow foam. The crude product was purified via reverse phaseHPLC on a C₁₈ column eluting starting with 60% acetonitrile/water. Afterconcentration, 870 mg (26%) of rapamycin-42-hemi-3'-oxoglutarate and 500mg (13%) of rapamycin-31,42-bishemi-3'oxoglutarate were isolated.

¹ H NMR (CDCl₃, 400 MHz) δ4.768 (m, 1H, CO₂ CH--CHOMe), 4.250 (m, 4H, O₂CCH₂ OCH₂ CO₂), 3.356 (s, 3H, CH₃ O--), 3.331 (s, 3H, CH₃ O--), 3.139(s, 3H, CH₃ O--), 1.759 (s, 3H, CH₃ C═C), 1.653 (s, 3H, CH₃ C═C); IR(KBr) 3420 (OH), 2920 (CH), 2875 (CH), 1740 (C═O), 1720 (C═O), 1640,1625, 1445, 1370, 1320, 1200, 1135, 1095, 980 cm⁻¹ ; MS (neg. ion FAB)1028 (M--H), 327, 167 (100), 148, 133, 115; High Res. MS (neg. ion FAB)Calcd for C₅₅ H₈₂ O₁₇ N (M--H) 1028.5597, Found 1028.5599.

Analysis Calcd for C₅₅ H₈₃ O₁₇ N.3H₂ O: C, 60.97; H, 8.22; N, 1.29.Found: C, 61.33; H, 7.74; N, 1.69.

The following representative compounds can be prepared from rapamycinand the appropriate half acid-ester by employing the method used toprepare the title compound in Example 7.

Rapamycin-42-hemi-3'-methylglutarate

Rapamycin-42-hemi-3',3'-dimethylglutarate

Rapamycin-42-hemi-3'-thioglutarate

Rapamycin-42-hemi-phthalate

Rapamycin-42-hemi-2',3'-pyridine dicarboxylate

EXAMPLE 8 Rapamycin-31,42-bishemi-3'-oxoglutarate

To a solution of 5.0 g (5.47 mmol) of rapamycin in 20 mL of drydichloromethane was added 3.17 g (27.3 mmol) of diglycolic anhydridefollowed by 2.17 mL (2.12 g, 27.3 mmol) of pyridine. To this was added400 mg of 4-dimethylaminopyridine and the reaction mixture was allowedto stir at reflux for 24 h. The solution was cooled to room temperature,poured into 2N HCl, and extracted three times with dichloromethane. Thecombined organic extracts were washed with brine, dried over anhydroussodium sulfate, decanted, and concentrated in vacuo to give a yellowfoam. The crude product was purified via reverse phase HPLC on a C₁₈column eluting starting with 60% acetonitrile/water. Afterconcentration, 1.75 g (28% ) of rapamycin-31,42-bishemi-3'-oxoglutaratewas isolated.

¹ H NMR (CDCl3, 400 MHz) δ4.785 (m, 1H, CO₂ CHCHOMe), 4.260 (m, 8H, O₂CCH₂ OCH₂ CO₂), 3.360 (s, 3H, CH₃ O--), 3.343 (s, 3H, CH₃ O--), 3.143(s, 3H, CH₃ O--), 1.775 (s, 3H, CH₃ C═C), 1.656 (s, 3H, CH₃ C═C); ¹³ CNMR (CDCl₃, MHz) 211.12 (C═O), 207.73 (C═O), 193.11 (C═O), 171.90 (C═O),171.59 (C═O), 170.15 (C═O), 169.35 (C═O), 168.83 (C═O), 166.63 (C═O); IR(KBr) 3420 (OH), 2920 (CH), 2850 (CH), 1740 (C═O), 1645, 1625, 1440,1370, 1190, 11300, 980 cm⁻¹ ; MS (neg. ion FAB) 1140 (M--H), 1122, 1026,990, 946, 913, 590, 475, 435, 321, 167, 148, 131 (100), 113; High Res.MS (neg. ion FAB) Calcd for C₅₉ H₈₆ O₂₁ N (M--H) 1144.5701, Found1144.5702.

Analysis Calcd for C₅₉ H₈₇ O₂₁ N: C, 61.82; H, 7.65; N, 1.22. Found: C,61.59; H, 7.36; N, 1.84.

EXAMPLE 9 Rapamycin-31,42-bishemi-3'-oxoglutarate disodium salt

Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (720 mg, 629 umol),prepared by the procedure employed in Example 8, was dissolved in 10 mLof 95% ethanol and 106 mg (1.26 mmol) of sodium bicarbonate was added.Water (1 mL) was added to completely dissolve the salt. Once dissolved,the light yellow solution was concentrated in vacuo to give a foamyyellow solid. The foam was dried in a drying pistol for 48 h, refluxingover dichloromethane at reduced pressure to give 435 mg (58%) of thedisodium salt.

¹ H NMR (d₆ -DMSO, 400 MHz) δ4.975 (m, 1H,--CHO₂ C), 4.593 (m, 1H,MeOCHCHO₂ C--), 4.135 (s, 2H, --O₂ CCH₂ OCH₂ CO₂ R), 3.617 (s,2H, --O₂CCH₂ OCH₂ CO₂ R), 3.299 (s, 6H, 2CH₃ O--), 3.232 (s, 3H, CH₃ O--), 1.614(s, 3H, CH₃ C═C), 1.553 (s, 3H, CH₃ C═C); IR (KBr) 3420 (OH), 2920 (CH),1735 (C═O), 1615, 1445, 1395, 1380, 1320, 1220, 1130, 1090, 980 cm⁻¹ ;

MS (neg. ion FAB) 1188 (M--1), 1166 (M--Na), 1144, 1051, 1028, 590, 459,167, 155 (100), 148, 133, 115.

Analysis Calcd for C₅₉ H₈₅ O₂₁ NNa₂.2H₂ O: C, 57.79; H, 7.26; N, 1.14.Found: C, 57.94; H, 7.11; N, 1.26.

EXAMPLE 10 Rapamycin-31,42-bishemi-3'-oxoglutarate bistromethamine salt

Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (1.01 g, 882 umol),prepared by the procedure employed in Example 8, was dissolved in 10 mLof 95% ethanol and 213 mg (1.76 mmol) of tris(hydroxymethyl)-methylamine was added. Water (1 mL) was added to completely dissolve theamine. Once dissolved, the yellow solution was concentrated in vacuo togive a foamy yellow solid. The very hygroscopic foam was dried in adrying pistol for 48 h, refluxing over dichloromethane at reducedpressure to give 805 mg (66%) of the bistromethamine salt.

¹ H NMR (d₆ -DMSO, 400 MHz) 5 4.955 (m, 1H, --CHO₂ C), 4.600 (m, 1H,MeOCHCHO₂ C--), 4.149 (s, 2H, --O₂ CCH₂ OCH₂ CO₂ R), 3.770 (s, 2H, --O₂CCH₂ OCH₂ CO₂ R), 3.407 (s, 6H, 2 CH₃ O--), 3.257 (s, 3H, CH₃ O--),1.806 (s, 3H, CH₃ C═C), 1.614 (s, 3H, CH₃ C═C); IR (KBr) 3400 (OH), 2920(CH), 1730 (C═O), 1620, 1550, 1450, 1395, 1370, 1200, 1060, 985 cm⁻¹ ;MS (neg. ion FAB) 1144 (M--H, free acid), 1028, 167, 148, 133 (100),115.

Analysis Calcd for C₆₇ H₁₀₉ O₂₇ N₃.H₂ O, C,57.22; H,7.90; N, 2.98.Found: C, 57.26; H, 7.90; N, 3.15.

EXAMPLE 11 Rapamycin-31,42-bishemisuccinate

To a solution of 2.0 g (2.2 mmol) of rapamycin in 10 mL of drydichloromethane was added 1.19 g (10.9 mmol) of succinic anhydridefollowed by 881 uL (861 mg, 10.9 mmol) of pyridine. To this was added200 mg of 4-dimethylaminopyridine and the reaction mixture refluxed for24 h. The solution was cooled to room temperature, poured into 2N HCl,and extracted three times with dichloromethane. The combined organicextracts were washed with brine, dried over anhydrous sodium sulfate,decanted, and concentrated in vacuo to give a yellow foam. The crudeproduct was purified via reverse phase HPLC on a C₁₈ column gradienteluting starting with 20% acetonitrile/water to 60% acetonitrile/water.Collected, after, concentration, 770 mg (31%) ofrapamycin-31,42-bishemisuccinate.

The purified bis-31,42 hemisuccinate of rapamycin (770 mg, 686 umol) wasdissolved in 10 mL of 95% ethanol and 166 mg (1.37 mmol) oftris(hydroxymethyl)methylamine was added. Water (1 mL) was added tocompletely dissolve the amine. Once dissolved, the yellow solution wasconcentrated in vacuo to give a foamy yellow solid. The very hygroscopicfoam was dried in a drying pistol for 24 h, refluxing over acetone atreduced pressure to give 890 mg (95%) of the bistromethamine salt. Thebistromethane salt was evaluated in the standard pharmacological testprocedures.

¹ H NMR (d₆ -DMSO, 400 MHz) 5.231 (m, 1H,--CHO₂ C), 4.554 (m, 1H,MeOCHCHO₂ C--), 3.426 (s, 6H, 2 CH₃ O--), 3.249 (s, 3H, CH₃ O--), 2.431(m, 8H, O₂ CCH₂ CH₂ CO₂ --), 1.700 (s, 3H, CH₃ C═C), 1.554 (s, 3H, CH₃C═C); ¹³ C NMR (d₆ -DMSO,) 211.28 (C═O), 205.23 (C═O), 199.59 (C═O),174.86 (C═O), 173.62 (C═O), 171.72 (C═O), 171.50 (C═O), 166.56 (C═O),166.53 (C═O); IR (KBr) 3420 (OH), 2940 (CH), 1735 (C═O), 1630, 1580,1460, 1400, 1380, 1170, 1070, 990 cm⁻¹ ; MS (neg. ion FAB) 1112 (M-1,free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100).

Analysis Calcd for C₆₇ H₁₀₉ O₂₅ N₃.2H₂ O: C, 57.80; H, 8.12; N, 3.01.Found: C, 57.91; H, 8.21, N, 2.37.

EXAMPLE 12 Rapamycin-42-monomethylsuccinate

To a solution of 5.0 g (5.47 mmol) of rapamycin, 2.17 g (8.2 mmol) ofmonomethylsuccinate, and 1.58 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20 mL ofdry dichoromethane was added 100 mg of 4-dimethylaminopyridine. Thesolution was strirred at room temperature overnight. The reactionmixture was poured into 2N aqueous HCl and extracted three times withethyl acetate. The organic layers were combined, washed with brine,dried over anhydrous sodium sulfate, filtered, and concentrated in vacuoto give a viscous yellow oil. Flash chromatography on a 60 mm×150 mmsilica column eluting with 20% ethyl acetate/hexane to 40% ethylacetate/hexane gave 2.35 g (42%) of purerapamycin-42-monomethylsuccinate.

¹ H NMR (CDCl₃, 400 MHz) δ4.79 (s, 1H,--OH), 4.69 (bm, 1H, 42--CHOC═O),3.69 (s, 3H, CH₃ OC═O), 3.38 (s, 3H, CH₃ O--), 3.33 (s, 3H, CH₃ O--),3.14 (s, 3H, CH₃ O--), 2.64 (m, 4H, O═CCH₂ CH₂ C═O); IR (KBr) 3440 (OH),2930 (CH), 1735 (C═O), 1720 (C═O), 1643, 1445, 1375, 1160, 1090, 990cm⁻¹ ; MS (neg. ion FAB) 1027 (M--), 590.

Analysis Calcd for C₅₆ H₈₅ NO₁₆.3H₂ O: C, 62.14; H, 8.48; N, 1.29.Found: C, 61.98; H, 7.60; N, 1.21.

EXAMPLE 13 Rapamycin-42-(4-trifluoromethylbenzyl)succinate

To a solution of 5.0 g (5.47 mmol) of rapamycin, 2.26 g (8.2 mmol) ofmono(4-trifluoromethylbenzyl)succinate, and 1.58 g of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20 mL ofdry dichoromethane was added 100 mg of 4-dimethylaminopyridine. Thesolution was strirred at room temperature overnight. The reactionmixture was poured into 2N aqueous HCl and extracted three times withethyl acetate. The organic layers were combined, washed with brine,dried over anhydrous sodium sulfate, filtered, and concentrated in vacuoto give a viscous yellow oil. Flash chromatography on a 60 mm×150 mmsilica column eluting with 10-20% ethyl acetate/hexane gave 365 mg (6%)of pure rapamycin-42-(4-trifluoromethylbenzyl)succinate.

¹ H NMR (CDCl₃, 400 MHz) δ7.46-7.63 (ab, 4H, arom), 5.19 (m, 2H, CH₂Ar), 4.79 (s, 1H, --OH), 4.68 (bm, 1H, 42-CHOC═O), 3.36 (s, 3H, CH₃O--), 3.33 (s, 3H, CH₃ O--), 3.14 (s, 3H, CH₃ O--), 2.71 (m, 4H, O═CCH₂CH₂ C═O); IR (KBr) 3445 (OH), 2940 (CH), 1740 (C═O), 1720 (C═O), 1650,1450, 1375, 1330, 1160, 1060, 990 cm⁻¹ ; MS (neg. ion FAB) 1171 (M--),1012, 590.

Analysis Calcd for C₆₃ H₈₈ NO₁₆ F₃.H₂ O: C, 63.56; H, 7.62; N, 1.18.Found: C, 63.34; H, 7.31; N, 1.28.

What is claimed is:
 1. A method of treating transplantation rejection ina mammal by administering an effective amount of a compound having thestructure ##STR10## whereinR¹, R², and R³ are each, independently,hydrogen or ##STR11## with the proviso that R¹, R², and R³ are not allhydrogen; R⁴ is --(CH₂)_(m) X(CH₂)_(n) CO₂ R⁵ or ##STR12## R⁵ and R⁶ areeach, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbonatoms, or phenyl which is optionally mono-, di-, or tri-substituted witha substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-15carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbonatoms, trifluoromethyl, amino, or a carboxylic acid;X is ##STR13## O, orS; R⁷ and R⁸ are each, independently, hydrogen or alkyl of 1-6 carbonatoms; Y is CH or N; m is 0-4; n is 0-4; with the proviso that m and nare not both 0 when X is O or S;or a pharmaceutically acceptable saltthereof.